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Expression and Purification of Active Recombinant Human Alpha-1 Antitrypsin (AAT) from Escherichia coli

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Alpha-1 Antitrypsin Deficiency

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1639))

Abstract

Well-established genetic manipulation procedures along with a fast doubling time, the ability to grow in inexpensive media, and easy scaleup make Escherichia coli (E. coli) a preferred recombinant protein expression platform. Human alpha-1 antitrypsin (AAT) and other serpins are easily expressed in E. coli despite their metastability and complicated topology. Serpins can be produced as soluble proteins or aggregates in inclusion bodies, and both forms can be purified to homogeneity. In this chapter, we describe an ion-exchange chromatography-based protocol that we have developed involving the use of two anion-exchange columns to purify untagged human AAT from E. coli. We also outline methods that can be used to determine the inhibitory activity of both AAT in cell lysates and purified AAT. Our protocol for the purification of bacterially expressed AAT yields pure and active protein at 6–7 mg/l culture.

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References

  1. Gettins PG (2002) Serpin structure, mechanism, and function. Chem Rev 102:4751–4804

    Article  CAS  PubMed  Google Scholar 

  2. Huntington JA, Carrell RW (2001) The serpins: nature's molecular mousetraps. Sci Prog 84:125–136

    Article  CAS  PubMed  Google Scholar 

  3. Huntington JA, Read RJ, Carrell RW (2000) Structure of a serpin-protease complex shows inhibition by deformation. Nature 407:923–926

    Article  CAS  PubMed  Google Scholar 

  4. Stratikos E, Gettins PG (1999) Formation of the covalents erpin-proteinase complex involves translocation of the proteinase by more than 70 Å and full insertion of the reactive center loop into beta-sheet A. Proc Natl Acad Sci U S A 96:4808–4813

    Google Scholar 

  5. Stockley RA (2014) Alpha1-antitrypsin review. Clin Chest Med 35:39–50

    Article  PubMed  Google Scholar 

  6. Gooptu B, Lomas DA (2009) Conformational pathology of the serpins: themes, variations, and therapeutic strategies. Annu Rev Biochem 78:147–176

    Article  CAS  PubMed  Google Scholar 

  7. Powell LM, Pain RH (1992) Effects of glycosylation on the folding and stability of human, recombinant and cleaved α1-antitrypsin. J Mol Biol 224:241–252

    Google Scholar 

  8. Sarkar A, Wintrode PL (2011) Effects of glycosylation on the stability and flexibility of a metastable protein: the human serpin α1-antitrypsin. Int J Mass Spectrom 302:69–75

    Google Scholar 

  9. Johansen H, Sutiphong J, Sathe G, Jacobs P, Cravado, A, Bollen A, Rosenberg M, Shatzman A (1987) High-level production of fully active human α1-antitrypsin in Escherichia coli, Mol Biol & Med 4:291–305.

    Google Scholar 

  10. Bischoff R, Speck D, Lepage P, Delatre L, Ledoux C, Brown SW, Roitsch C (1991) Purification and biochemical characterization of recombinant α1-antitrypsin variants expressed in Escherichia coli. Biochemistry 30:3464–3472

    Article  CAS  PubMed  Google Scholar 

  11. Hopkins PC, Carrell RW, Stone SR (1993) Effects of mutations in the hinge region of serpins. Biochemistry 32:7650–7657

    Article  CAS  PubMed  Google Scholar 

  12. Griffiths SW, Cooney CL (2002) Development of a peptide mapping procedure to identify and quantify methionine oxidation in recombinant human α1-antitrypsin. J Chromat A 942:133–143

    Article  CAS  Google Scholar 

  13. Agarwal S, Jha S, Sanyal I, Amla DV (2010) Expression and purification of recombinant human α1-proteinase inhibitor and its single amino acid substituted variants in Escherichia coli for enhanced stability and biological activity. J Biotech 147:64–72

    Article  CAS  Google Scholar 

  14. Zhou A, Carrell RW, Huntington JA (2001) The serpin inhibitory mechanism is critically dependent on the length of the reactive center loop. J Biol Chem 276:27541–27547

    Article  CAS  PubMed  Google Scholar 

  15. Pearce MC, Cabrita LD (2011) Production of recombinant serpins in Escherichia coli. Methods Enzymol 501:13–28

    Article  CAS  PubMed  Google Scholar 

  16. Krishnan B, Gierasch LM (2011) Dynamic local unfolding in the serpin α-1 antitrypsin provides a mechanism for loop insertion and polymerization. Nat Struct Mol Biol 18:222–226

    Google Scholar 

  17. Kwon KS, Lee S, Yu MH (1995) Refolding of α1-antitrypsin expressed as inclusion bodies in Escherichia coli: characterization of aggregation. Biochim Biophys Acta 1247:179–184

    Article  PubMed  Google Scholar 

  18. Bottomley SP, Stone SR (1998) Protein engineering of chimeric Serpins: an investigation into effects of the serpin scaffold and reactive centre loop length. Protein Eng 11:1243–1247

    Article  CAS  PubMed  Google Scholar 

  19. Peterson FC, Gordon NC, Gettins PG (2000) Formation of a noncovalent serpin-proteinase complex involves no conformational change in the serpin. Use of 1H-15NHSQC NMR as a sensitive nonperturbing monitor of conformation. Biochemistry 39:11884–11892

    Google Scholar 

  20. Dementiev A, Dobo J, Gettins PG (2006) Active site distortion is sufficient for proteinase inhibition by serpins: structure of the covalent complex of α1-proteinase inhibitor with porcine pancreatic elastase. J Biol Chem 281:3452–3457

    Google Scholar 

  21. Huber R, Carrell RW (1989) Implications of the three-dimensional structure of α1-antitrypsin for structure and function of serpins. Biochemistry 28:8951–8966

    Google Scholar 

  22. UniProt Consortium (2015) UniProt: a hub for protein information. Nucleic Acids Res 43:D204–D212

    Google Scholar 

  23. Laska, M. E. (2001) The effect of dissolved oxygen on recombinant protein degradation in Escherichia coli, Thesis, Massachusetts Institute of Technology, Cambridge, MA

    Google Scholar 

  24. Pannell R, Johnson D, Travis J (1974) Isolation and properties of human plasma α-1-proteinase inhibitor. Biochemistry 13:5439–5445

    Google Scholar 

  25. James HL, Cohen AB (1978) Mechanism of inhibition of porcine elastase by human alpha-1-antitrypsin. J Clin Invest 62:1344–1353

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Seo EJ, Im H, Maeng JS, Kim KE, Yu MH (2000) Distribution of the native strain in human α1-antitrypsin and its association with protease inhibitor function. J Biol Chem 275:16904–16909

    Article  CAS  PubMed  Google Scholar 

  27. Dolmer K, Gettins PG (2012) How the serpin α1-proteinase inhibitor folds. J Biol Chem 287:12425–12432

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Chase T Jr, Shaw E (1967) P-Nitrophenyl-p'-guanidinobenzoate HCl: a new active site titrant for trypsin. Biochem Biophys Res Comm 29:508–514

    Article  CAS  PubMed  Google Scholar 

  29. Erlanger BF, Kokowsky N, Cohen W (1961) The preparation and properties of two new chromogenic substrates of trypsin. Arch Biochem Biophys 95:271–278

    Article  CAS  PubMed  Google Scholar 

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Acknowledgment

We thank Prof. C. L. Cooney (Massachusetts Institute of Technology) for providing the pEAT8-137 plasmid, and Prof. M. H. Yu (Korea Institute of Science and Technology), who provided the original source of the pEAT8 plasmid. This work was supported by grants from the Alpha-1 Foundation and DBT-Ramalingaswami Re-entry Fellowship (to B.K.), the US National Institutes of Health, OD-00045 (to L.M.G.), and R01 GM094848 (to A.G., D.N.H. & L.M.G.). B.K. thanks CSIR-IMTECH for the protein instrumentation facility.

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Authors and Affiliations

  1. G.N. Ramachandran Protein Centre, CSIR–Institute of Microbial Technology, Sector 39-A, Chandigarh, 160 036, India

    Beena Krishnan

  2. Department of Biology, Brandeis University Waltham, Waltham, MA, USA

    Lizbeth Hedstrom

  3. Department of Chemistry, Brandeis University Waltham, Waltham, MA, USA

    Lizbeth Hedstrom

  4. Department of Biochemistry and Molecular Biology, University of Massachusetts Amherst, Amherst, MA, USA

    Daniel N. Hebert, Lila M. Gierasch & Anne Gershenson

  5. Department of Chemistry, University of Massachusetts Amherst, Amherst, MA, USA

    Lila M. Gierasch

Authors
  1. Beena Krishnan
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  2. Lizbeth Hedstrom
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  3. Daniel N. Hebert
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  4. Lila M. Gierasch
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  5. Anne Gershenson
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Corresponding author

Correspondence to Beena Krishnan .

Editor information

Editors and Affiliations

  1. Horae Gene Therapy Center, University of Massachusetts Medical School, Worcester, Massachusetts, USA

    Florie Borel

  2. Horae Gene Therapy Center, University of Massachusetts Medical School, Worcester, Massachusetts, USA

    Christian Mueller

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Krishnan, B., Hedstrom, L., Hebert, D.N., Gierasch, L.M., Gershenson, A. (2017). Expression and Purification of Active Recombinant Human Alpha-1 Antitrypsin (AAT) from Escherichia coli . In: Borel, F., Mueller, C. (eds) Alpha-1 Antitrypsin Deficiency . Methods in Molecular Biology, vol 1639. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7163-3_19

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  • DOI: https://doi.org/10.1007/978-1-4939-7163-3_19

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7161-9

  • Online ISBN: 978-1-4939-7163-3

  • eBook Packages: Springer Protocols

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